Novel PPOX mutations in two variegate porphyria pedigrees

Novel PPOX mutations in two variegate porphyria pedigrees | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel PPOX mutations in two variegate porphyria pedigrees Shubin Lei, Lu Yang, Ruixuan Zhang, Xin Guan, Xiuli Zhao, Tao Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3330892/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Variegate porphyria (VP) is an autosomal dominant inherited disease caused by mutation of the PPOX gene and impacted activity of the protoporphyrinogen oxidase (PPOX). We report two pedigrees of young women who present VP-related manifestation such as blistering, flushing, scarring, and hyperpigmentation after sunlight exposure. The genetic analysis of PPOX gene was performed. Methods Whole exome sequencing was conducted for both probands, followed by poly chain reaction (PCR) to amplify interested region and validate the suspicious mutations. Reverse transcriptive PCR was conducted to explore the impact of intronic mutation on the splicing process. Results The genetic analysis revealed two novel mutations: c.82_83delCC:p.Pro28* and c.222 + 2delT in heterozygous state. The first mutation leads to a premature termination of PPOX translation and the second one causes the insertion of intron2 between exon2 and exon3. Both of the probands inherit PPOX mutation from their healthy father. Conclusions Our study provides molecular diagnosis for two VP pedigrees and identified two novel PPOX mutations. We propose a hypothesis that the incomplete penetrance of VP in these two cases might be associated with sexuality and hormone level. variegate porphyria mutation protoporphyrinogen oxidase Figures Figure 1 Figure 2 Figure 3 Introduction Porphyria is a group of metabolic diseases caused by the change of enzyme activity in the pathway of heme synthesis. Synthesis of heme involves 8 enzymes encoded by different genes. The mutation of these genes could impact the activity of counterpart enzymes and lead to different types of porphyria. According to clinical features, porphyria could be categorized into 3 subgroups: acute, cutaneous and mixed type of porphyria( 1 ). Acute porphyria includes delta-aminolevulinic acid dehydratase porphyria (ADP) and acute intermittent porphyria (AIP). The main clinical manifestation of acute porphyria is neurovisceral crises such as abdominal pain, peripheral neuropathy of motor and sensory system, and acute vesicant skin lesion. Cutaneous porphyria is characterized as skin photosensitivity and increased skin fragility, which includes porphyria cutanea tarda (PCT), hepatoerythropoietic porphyria (HEP), congenital erythropoietic porphyria (CEP), erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP). Mixed porphyria includes hereditary coproporphyria (HCP) and variegate porphyria (VP) and patients could have all symptoms mentioned above. VP (OMIM:176200), as one of the mixed types of porphyria, is an autosomal dominant (AD) inherited disease caused by mutation of the PPOX gene (reference sequenceNM_000309.5) located at 1q23.3 on human chromatin. PPOX is composed of 12 exons spread over 12kb DNA, encoding the penultimate enzyme for heme synthesis: protoporphyrinogen oxidase (PPOX)( 2 ). The incidence of VP is 3.2/1000000 in Europe（3）; while the rate could reach 1/300 among South African with Holland descent（2）. Clinical manifestation of VP could be highly heterogeneous. Some patients could have severe neurovisceral crises or chronic photosensitivity or non-blistering skin lesion, while others could have no obvious symptoms other than subclinical biochemical changes. Previous researches showed that approximately 60% of the VP patients only had skin symptoms, such as erythema and erosive photodermatoses; while 20% of the patients only had acute neurovisceral attacks and rest had both manifestations of acute and cutaneous porphyria（4）. Except for the typical presentation of porphyria, some malignant diseases have been reported to be associated with VP, including hepatocellular carcinoma（5）. Although VP could greatly impact patients’ life quality, and even be lethal sometimes, its pathogenesis is still not fully understood, especially regarding the diversity of clinical presentation and disease penetrance. In this study, we reported two VP pedigrees with two novel heterozygous PPOX mutations inherited in autosomal dominant pattern with incomplete penetrance: c.82_83delCC:p.Pro28* and c.222 + 2delT. We also discussed the possible mechanisms and contributing factors to disease onset. Our study expanded the mutation pool of PPOX gene, which would be of great value for disease diagnosis, prevention and genetic counseling. Materials and methods Ethical Considerations Medical records were collected and clinical photographs were taken for analysis. Peripheral blood was collected for genetic analysis. Written informed consents were obtained from each participant in line with the Declaration of Helsinki. This study was approved by Institutional Review Board (IRB) of the research institution(No.I-22PJ622). Informed consents have been acquired for the publication of clinical manifestation. Isolation of genomic DNA and RNA Peripheral blood samples were obtained from all participants. Genomic DNA was extracted from the peripheral blood samples by the standard method of proteinase K and phenol/chloroform extraction( 6 ). RNA was extracted from peripheral blood of proband II-1 using TRIzol method（7）. Whole-exome sequencing Approximate 500µl peripheral blood of each proband and participant was used for whole-exome sequencing. The exome was captured using an Agilent Sure Select Human All Exon kit (Agilent Technologies, Wilmington, DE), according to the manufacturer’s instructions. DNA was broken into fragments of 180– 280bp using an ultrasonoscope. Sequencing results were then aligned to the human reference genome sequence of the UCSC database. Variants were filtered by the Single Nucleotide Polymorphism Database and the 1000 Genomes database. Validation of mutations Poly chain reaction (PCR) amplification of PPOX gene was conducted to validate the variants reported by whole-exome sequencing. Approximate 500bp DNA sequence around variant site (including exon1, intron1 and part of exon2) in pedigree1 and 300bp DNA sequence around variant site of pedigree2(including exon2 and majority of intron2) was amplified. For splicing variant identified in pedigree2, reverse transcriptive PCR (rtPCR) was conducted with Prime Script™ RT reagent Kit of Takara to get cDNA, followed by amplification of exon2 and exon3 based on cDNA. Result Clinical manifestation Two pedigrees of VP were incorporated (Fig. 1 ). Careful physical examinations were performed for each patient and the clinical records have been analyzed. Pedigree 1 Proband in pedigree 1 is a 30-year-old woman complaining of recurrent blistering, scarring, and sclerosis in her face and biliteral upper limb (Fig. 1 . a-c). She has been complaining about the symptoms and the lesions would be slightly exaggerated by sunlight exposure. Lab examination showed abnormal liver function with increased level of aspartate aminotransferase (88 U/L, reference range:5–35 U/L), direct bilirubin (13.7µmol/L, reference range: 0–5µmol/L), total bile acid (26.8µmol/L, reference range: 0–10µmol/L), and slightly decreased albumin level (33.4g/L, reference range: 39.7–49.4 g/L). Although none of her family members had porphyria-related clinical manifestation, the patient was highly suspected of porphyria and genetic analysis was conducted for each family members for disease diagnosis and genetic counselling. Pedigree 2 Proband in pedigree 2 is a 30-years-old woman with four-year disease course. She had burning erythema and blisters on her face, neck and bilateral hand backs, with relief after sunlight avoidance (Fig. 1 . d-g). The symptoms were more severe in summer. The urine sample of the patient yielded pink fluorescent under Wood’s lamp (Fig. 1 . e). The patient was diagnosed as porphyria and suggested with light shielding. She received further genetic analysis because of the exaggerations of her skin symptoms in the past year. None of her family members had porphyria-related clinical manifestation, such as photodermatoses or acute neurovisceral attack. Genetic analysis Whole-exome sequencing Whole-exome sequencing of Pedigree 1 identified a novel deletion mutation with high pathogenicity: c.82_83delCC:p.Pro28* in proband 1 and her father; while sequencing in Pedigree 2 identified a intronic mutation of c.222 + 2delT in proband 2 and her father (Table 1 ). The pathogenicity was accessed according to guidance of The American College of Medical Genetics and Genomics (ACMG)( 8 ). Table 1 result of whole exome sequencing Pedigree Variant Mutation type Position Zygote type Pathogenicity Proband Father Mother Pedigree 1 c.82_83delCC:p.Pro28* Small deletion 161136714 Heterozygote Heterozygote WT Extremely highly pathogenic Pedigree 2 c.222 + 2delT Splicin g mutation 161137025 Heterozygote Heterozygote WT Extremely highly pathogenic Identification of a nonsense variant in PPOX in Pedigree 1 Sanger sequencing of the proband and her father indicated the presence of a heterozygous nonsense variant c.82_83delCC:p.Pro28* in PPOX . This variant led to the substitution of codon CCU coding for proline to a terminal codon at position 28 of the amino acid sequence (Fig. 2 ). Identification of an intronic variant in PPOX in Pedigree 2 Sanger sequencing of the proband and her father indicated the presence of a heterozygous intronic variant c.222 + 2delT in PPOX . Because the position is very close to exon 2, it may lead to a different protein sequence by impacting the process of splicing. In order to verify the impact of variant c.222 + 2delT on splicing, rtPCR was conducted based on RNA of the proband 2, followed by amplification and Sanger sequencing of the exon 2 and exon 3 of the acquired cDNA. The result indicated an insertion of intron2 between these two exons (Fig. 3 ). Pathogenicity analysis To explore the conservatism of the mutation site in Pedigree 1, a conservation analysis was conducted by comparing protein sequence among different species, including human, mouse and cat. The result showed that the mutation site was highly conservative between different species (Fig. 2 . b). Protein structure of normal and mutated PPOX were predicted and the result showed that the small deletion in Pedigree 1 (c.82_83delCC) led to a premature termination of the transcription(Fig. 2 . c-d) and that the splicing mutation in Pedigree 2 (c.222 + 2delT) could cause the insertion of 45 amino acids (Fig. 3 . d-e) between exon2 and exon3. Discussion This research identified two novel mutations of PPOX . Variant in pedigree1 was a nonsense mutation(c.82_83delCC:p.Pro28*), leading to premature termination of the transcription and abnormal protein structure. Although variant at the site of first pedigree has not been reported before, previous research has identified p.Cys26* in VP pedigree( 9 ), which is close to the mutation in pedigree 1 reported in this study. Apart from that, a frameshift variant c.78insC near the mutation site in pedigree 1 has been reported to be able to cut down the relative PPOX activity to 0% compared to wildtype in both eukaryotic and procaryotic expression systems （10）. Variant in pedigree 2 (c.222 + 2delT ) was a typical splicing mutation located at a highly conservative site in intron 2 of PPOX gene. The variant could be pathogenic by impacting the process of normal splicing and protein structure and function. It is noteworthy that the in spite of the similar disease course, proband 1 tended to have more obvious hyperpigmentation and scar formation than proband 2. Furthermore, the liver function of proband 1 was extensively impaired, indicating a likely more severe phenotype associated with the nonsense mutation(c.82_83delCC:p.Pro28*), probably due to its huge impact on the protein structure(Fig. 2 . c-d). However, her father carrying the same mutation did not suffer from any porphyria attack, indicating the onset of the disease is not solely dependent on hereditary factors. In fact, VP has been reported to be an autosomal dominant inherited disease with low penetrance. Clinical presentation of VP varies from no overt clinical symptoms, chronic photosensitivity to acute neurovisceral crises. Penetrance of 40% and 50% has been reported in two pedigrees of VP carrying variant p. R59W and IVS6 + 2T > A, respectively ( 11 , 12 ). Haplogroup H of mtDNA is also associated with the penetrance（12）. Up to now, over 180 mutations of VP have been identified （13）. It has been reported that among the three most common mutations in Finnish VP patients, the missense variant I12T might be an indicator for milder symptom （14）, however, there is no clear correlation between genotype and phenotype according to most of the previous publication（15, 16）. Even among individuals with the same variant, clinical manifestation could be different （17）, as in our presenting pedigrees. Of the eight porphyria, five are inherited through an autosomal dominant pattern. In addition to VP, the clinical penetrance of other four AD porphyria is also low. Porphyria attacks might be induced by factors which either lead to increasing demand for heme or an additional decrease in enzyme activity( 18 ). These non-hereditary factors include medications, infections, dieting, alcohol, hormones and neuropsychiatric factors（13）. It has been reported that exogenous oestrogen is associated with the onset of PCT （18）. Some scholars also believe that acute hepatic porphyria could be triggered by various hormonal changes, such as the use of high progesterone birth control medications（19, 20）. Furthermore, in AIP and VP, some of the acute attacks are related to menstruation, which could probably be reversed by Gonadotropin-releasing hormone (GnRH) agonists（20, 21）. Recently, a retrospective study has evaluated the penetrance of VP in 23 genetically heterogenous Finnish families and found that penetrance was higher among female than male (44% vs. 33%), and that during reproductive years(12–49 years of age), the risk for acute symptoms is at the highest, reenforcing that hormone might be a contributing factor in the pathogenesis of the disease（22）.In this study, both female probands were heterozygotes who inherited their mutations from their heterozygous fathers who had not suffered from any symptoms related to VP yet. Besides, they both have overt clinical manifestation in their reproductive years. This could be possibly explained by their sexuality and hormone levels as indicated in previous publication. In this study, we identified two novel mutations of PPOX gene: c.82_83delCC:p.Pro28* and c.222 + 2delT, which cause abnormal structure and function of the protein. The penetrance of these two mutations might be associated with sexuality. These findings expanded the genotypic and phenotypic spectrum of VP, which would be of critical value for disease diagnosis, genetic counseling and patient management. Declarations Funding This work was supported by the National High Level Hospital Clinical Research Funding (No. 2022-PUMCH-A-164 to T.W), the National College Students Innovation and Entrepreneurship Training Program (No.202010023059 to S.L,X.Z), the Beijing Natural Science Foundation (No. Z210017 to T.W) and Peking Union Medical College Hospital (No. ZC201911051 to T.W). References D M Bissell, K E Anderson and H L Bonkovsky Porphyria. N Engl J Med 2017;377:862-872. R E Kirsch, P N Meissner and R J Hift Variegate porphyria. Semin Liver Dis 1998;18:33-41. P Mustajoki Variegate porphyria. Twelve years' experience in Finland. Q J Med 1980;49:191-203. Z Karim, S Lyoumi, G Nicolas , et al. Porphyrias: A 2015 update. Clin Res Hepatol Gastroenterol 2015;39:412-25. A Luvai, W Mbagaya, D Narayanan , et al. Hepatocellular carcinoma in variegate porphyria: a case report and literature review. Ann Clin Biochem 2015;52:407-12. A Manzin, G Salvoni, P Bagnarelli , et al. A single-step DNA extraction procedure for the detection of serum hepatitis B virus sequences by the polymerase chain reaction. J Virol Methods 1991;32:245-53. D C Rio, M Ares, Jr., G J Hannon , et al. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc 2010;2010:pdb.prot5439. S Richards, N Aziz, S Bale , et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405-24. S D Whatley, N G Mason, J R Woolf , et al. Diagnostic strategies for autosomal dominant acute porphyrias: retrospective analysis of 467 unrelated patients referred for mutational analysis of the HMBS, CPOX, or PPOX gene. Clin Chem 2009;55:1406-14. M von und zu Fraunberg, R Tenhunen and R Kauppinen Expression and characterization of six mutations in the protoporphyrinogen oxidase gene among Finnish variegate porphyria patients. Mol Med 2001;7:320-8. R J Hift, D Meissner and P N Meissner A systematic study of the clinical and biochemical expression of variegate porphyria in a large South African family. Br J Dermatol 2004;151:465-71. A Bonnin, A Picornell, J Orfila , et al. Clinic and genetic evaluation of variegate porphyria (VP) in a large family from the Balearic Islands. J Inherit Metab Dis 2009;32 Suppl 1: Z Novakova, J Mikesova, M Ondrakova , et al. Molecular characterization of a novel His333Arg variant of human protoporphyrinogen oxidase IX. Biochem Biophys Res Commun 2022;588:182-186. M von und zu Fraunberg, K Timonen, P Mustajoki , et al. Clinical and biochemical characteristics and genotype-phenotype correlation in Finnish variegate porphyria patients. Eur J Hum Genet 2002;10:649-57. S D Whatley, H Puy, R R Morgan , et al. Variegate porphyria in Western Europe: identification of PPOX gene mutations in 104 families, extent of allelic heterogeneity, and absence of correlation between phenotype and type of mutation. Am J Hum Genet 1999;65:984-94. E Di Pierro, P Ventura, V Brancaleoni , et al. Clinical, biochemical and genetic characteristics of Variegate Porphyria in Italy. Cell Mol Biol (Noisy-le-grand) 2009;55:79-88. M Lecha, C Badenas, S Puig , et al. Genetic studies in variegate porphyria in Spain. Identification of gene mutations and family study for carrier detection. J Eur Acad Dermatol Venereol 2006;20:974-9. M N Badminton and G H Elder Molecular mechanisms of dominant expression in porphyria. J Inherit Metab Dis 2005;28:277-86. M Yasuda, B Chen and R J Desnick Recent advances on porphyria genetics: Inheritance, penetrance & molecular heterogeneity, including new modifying/causative genes. Mol Genet Metab 2019;128:320-331. R J Hift and P N Meissner An analysis of 112 acute porphyric attacks in Cape Town, South Africa: Evidence that acute intermittent porphyria and variegate porphyria differ in susceptibility and severity. Medicine (Baltimore) 2005;84:48-60. E Innala, T Backstrom, M Bixo , et al. Evaluation of gonadotropin-releasing hormone agonist treatment for prevention of menstrual-related attacks in acute porphyria. Acta Obstet Gynecol Scand 2010;89:95-100. K Baumann and R Kauppinen Penetrance and predictive value of genetic screening in acute porphyria. Mol Genet Metab 2020;130:87-99. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3330892","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":231455073,"identity":"347fa6b5-90cf-4756-9414-4ed08eeb5b8a","order_by":0,"name":"Shubin Lei","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shubin","middleName":"","lastName":"Lei","suffix":""},{"id":231455074,"identity":"acbd1e04-d5f0-4999-b50a-e24c265f9439","order_by":1,"name":"Lu Yang","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Yang","suffix":""},{"id":231455075,"identity":"c2f5b335-a1bf-461c-880c-88a3cac5f511","order_by":2,"name":"Ruixuan Zhang","email":"","orcid":"","institution":"Peking Union Medical College Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruixuan","middleName":"","lastName":"Zhang","suffix":""},{"id":231455076,"identity":"70530b48-47d3-4f3c-aebd-4be003b8bcad","order_by":3,"name":"Xin Guan","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Guan","suffix":""},{"id":231455077,"identity":"1932f74b-6335-4a84-b41d-42377f70169e","order_by":4,"name":"Xiuli Zhao","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiuli","middleName":"","lastName":"Zhao","suffix":""},{"id":231455078,"identity":"55947e52-bf75-4e07-8d55-7368d73d9587","order_by":5,"name":"Tao Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYJADNoYPBjZyxKk9ANXCOKMgzZg0Lcw8Hw4nElRtcPzs4dcfamzyDK4dfvbYxoA5gYH98NENeLWcyUuzOHAsrdjgdpq5cY4BWx4DT1raDXxazA7kmBkcYDucuOF2Dpt0jgFPMYMEjxl+LeffALX8g2qxMJBIbCCo5UaO8YODbVAtDAYGhLXY33hjxnC2L61Y8naamWSPQYIxGyG/SPbnGH+o+GaTx3c7+ZnEjz//5fjZDx/DqwUI2CSARAKCS0A5CDB/QNEyCkbBKBgFowAdAAD1l06Nt1vmFwAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2023-09-06 10:59:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3330892/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3330892/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":43005983,"identity":"c5666b1d-7ec2-41bd-8416-a396a1a3d04c","added_by":"auto","created_at":"2023-09-12 14:08:55","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":143996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical manifestations and pedigrees of the two probands in Pedigree 1\u003c/strong\u003e \u003cstrong\u003e(a-c)\u003c/strong\u003e and Pedigree 2\u003cstrong\u003e (d-g)\u003c/strong\u003e. \u003cstrong\u003e(a)\u003c/strong\u003e. blistering, ulceration and scaring on bilateral hand backs. (\u003cstrong\u003eb). \u003c/strong\u003eskin rash, scaring and hyperpigmentation over the face. (\u003cstrong\u003ec)\u003c/strong\u003e. pedigree of proband 1. \u003cstrong\u003ed-e.\u003c/strong\u003e erythema, flushing and mild ulceration on face and neck. \u003cstrong\u003ef\u003c/strong\u003e. pink fluorescence under Wood’s lamp of the urine sample. \u003cstrong\u003eg\u003c/strong\u003e. pedigree of proband 2. Closed circle and square affected female and male with clinically symptomatic disease; Open circle and square unaffected family member; half-filled square asymptomatic individual carrying the affected gene. Arrows: probands.\u003c/p\u003e","description":"","filename":"figure1v3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3330892/v1/bc30ff32b1c5ba415db46645.jpeg"},{"id":43005980,"identity":"19c4639d-4416-4438-9c4d-7afb77c0bb47","added_by":"auto","created_at":"2023-09-12 14:08:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequencing results indicate the heterozygous variant c.82_83delCC of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePPOX\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in Pedigree1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea)\u003c/strong\u003e: In Pedigree 1, I-2 and II-1 were heterozygous of c.82_83delCC (p. Pro28*) in \u003cem\u003ePPOX\u003c/em\u003e. (\u003cstrong\u003eb)\u003c/strong\u003e: Sequence alignment of the nonsense variants in \u003cem\u003ePPOX\u003c/em\u003e was analyzed by comparing the amino acid among different sequences. \u003cstrong\u003ec-d\u003c/strong\u003e: Overall structure of the normal PPOX protein\u003cstrong\u003e(c) \u003c/strong\u003eand p. Pro28*mutant protein\u003cstrong\u003e(d).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure2v3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3330892/v1/530fdcae395abb4686d5af8f.jpeg"},{"id":43008226,"identity":"36bea81d-e84d-40ce-97a5-0c821a83538c","added_by":"auto","created_at":"2023-09-12 14:16:55","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequencing results indicate the heterozygous variant c.222+2delT of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePPOX\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in Pedigree2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea)\u003c/strong\u003e: In Pedigree 2, I-2 and II-1 were heterozygous of c.222+2delT in \u003cem\u003ePPOX\u003c/em\u003e (b): intron2 was inserted between exon2 and exon3 \u003cstrong\u003e(c)\u003c/strong\u003e: ideograph of aberrant splicing caused by variant c.222+2delT \u003cstrong\u003ed-e\u003c/strong\u003e: Overall structure of the normal PPOX protein\u003cstrong\u003e(d)\u003c/strong\u003e and c.222+2delT mutant protein red line: inserted amino acids\u003cstrong\u003e(e).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure3v3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3330892/v1/dfbbb4e35fa28a19fdda49e5.jpeg"},{"id":49149911,"identity":"4636594a-5db2-438c-83b6-fb94ea7df626","added_by":"auto","created_at":"2024-01-03 23:37:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":582562,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3330892/v1/d797a685-dcd3-4fee-bcb1-03fd6a833b31.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel PPOX mutations in two variegate porphyria pedigrees","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePorphyria is a group of metabolic diseases caused by the change of enzyme activity in the pathway of heme synthesis. Synthesis of heme involves 8 enzymes encoded by different genes. The mutation of these genes could impact the activity of counterpart enzymes and lead to different types of porphyria. According to clinical features, porphyria could be categorized into 3 subgroups: acute, cutaneous and mixed type of porphyria(\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e). Acute porphyria includes delta-aminolevulinic acid dehydratase porphyria (ADP) and acute intermittent porphyria (AIP). The main clinical manifestation of acute porphyria is neurovisceral crises such as abdominal pain, peripheral neuropathy of motor and sensory system, and acute vesicant skin lesion. Cutaneous porphyria is characterized as skin photosensitivity and increased skin fragility, which includes porphyria cutanea tarda (PCT), hepatoerythropoietic porphyria (HEP), congenital erythropoietic porphyria (CEP), erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP). Mixed porphyria includes hereditary coproporphyria (HCP) and variegate porphyria (VP) and patients could have all symptoms mentioned above.\u003c/p\u003e\n\u003cp\u003eVP (OMIM:176200), as one of the mixed types of porphyria, is an autosomal dominant (AD) inherited disease caused by mutation of the \u003cem\u003ePPOX\u003c/em\u003e gene (reference sequenceNM_000309.5) located at 1q23.3 on human chromatin. \u003cem\u003ePPOX\u003c/em\u003e is composed of 12 exons spread over 12kb DNA, encoding the penultimate enzyme for heme synthesis: protoporphyrinogen oxidase (PPOX)(\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e). The incidence of VP is 3.2/1000000 in Europe（3）; while the rate could reach 1/300 among South African with Holland descent（2）. Clinical manifestation of VP could be highly heterogeneous. Some patients could have severe neurovisceral crises or chronic photosensitivity or non-blistering skin lesion, while others could have no obvious symptoms other than subclinical biochemical changes. Previous researches showed that approximately 60% of the VP patients only had skin symptoms, such as erythema and erosive photodermatoses; while 20% of the patients only had acute neurovisceral attacks and rest had both manifestations of acute and cutaneous porphyria（4）. Except for the typical presentation of porphyria, some malignant diseases have been reported to be associated with VP, including hepatocellular carcinoma（5）.\u003c/p\u003e\n\u003cp\u003eAlthough VP could greatly impact patients\u0026rsquo; life quality, and even be lethal sometimes, its pathogenesis is still not fully understood, especially regarding the diversity of clinical presentation and disease penetrance. In this study, we reported two VP pedigrees with two novel heterozygous \u003cem\u003ePPOX\u003c/em\u003e mutations inherited in autosomal dominant pattern with incomplete penetrance: c.82_83delCC:p.Pro28* and c.222\u0026thinsp;+\u0026thinsp;2delT. We also discussed the possible mechanisms and contributing factors to disease onset. Our study expanded the mutation pool of \u003cem\u003ePPOX\u003c/em\u003e gene, which would be of great value for disease diagnosis, prevention and genetic counseling.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eEthical Considerations\u003c/h2\u003e\n\u003cp\u003eMedical records were collected and clinical photographs were taken for analysis. Peripheral blood was collected for genetic analysis. Written informed consents were obtained from each participant in line with the Declaration of Helsinki. This study was approved by Institutional Review Board (IRB) of the research institution(No.I-22PJ622). Informed consents have been acquired for the publication of clinical manifestation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eIsolation of genomic DNA and RNA\u003c/h2\u003e\n\u003cp\u003ePeripheral blood samples were obtained from all participants. Genomic DNA was extracted from the peripheral blood samples by the standard method of proteinase K and phenol/chloroform extraction(\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e). RNA was extracted from peripheral blood of proband II-1 using TRIzol method（7）.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eWhole-exome sequencing\u003c/h2\u003e\n\u003cp\u003eApproximate 500\u0026micro;l peripheral blood of each proband and participant was used for whole-exome sequencing. The exome was captured using an Agilent Sure Select Human All Exon kit (Agilent Technologies, Wilmington, DE), according to the manufacturer\u0026rsquo;s instructions. DNA was broken into fragments of 180\u0026ndash; 280bp using an ultrasonoscope. Sequencing results were then aligned to the human reference genome sequence of the UCSC database. Variants were filtered by the Single Nucleotide Polymorphism Database and the 1000 Genomes database.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eValidation of mutations\u003c/h2\u003e\n\u003cp\u003ePoly chain reaction (PCR) amplification of \u003cem\u003ePPOX\u003c/em\u003e gene was conducted to validate the variants reported by whole-exome sequencing. Approximate 500bp DNA sequence around variant site (including exon1, intron1 and part of exon2) in pedigree1 and 300bp DNA sequence around variant site of pedigree2(including exon2 and majority of intron2) was amplified. For splicing variant identified in pedigree2, reverse transcriptive PCR (rtPCR) was conducted with Prime Script\u0026trade; RT reagent Kit of Takara to get cDNA, followed by amplification of exon2 and exon3 based on cDNA.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eClinical manifestation\u003c/h2\u003e\n\u003cp\u003eTwo pedigrees of VP were incorporated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Careful physical examinations were performed for each patient and the clinical records have been analyzed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003ePedigree 1\u003c/h2\u003e\n\u003cp\u003eProband in pedigree 1 is a 30-year-old woman complaining of recurrent blistering, scarring, and sclerosis in her face and biliteral upper limb (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. a-c). She has been complaining about the symptoms and the lesions would be slightly exaggerated by sunlight exposure. Lab examination showed abnormal liver function with increased level of aspartate aminotransferase (88 U/L, reference range:5\u0026ndash;35 U/L), direct bilirubin (13.7\u0026micro;mol/L, reference range: 0\u0026ndash;5\u0026micro;mol/L), total bile acid (26.8\u0026micro;mol/L, reference range: 0\u0026ndash;10\u0026micro;mol/L), and slightly decreased albumin level (33.4g/L, reference range: 39.7\u0026ndash;49.4 g/L). Although none of her family members had porphyria-related clinical manifestation, the patient was highly suspected of porphyria and genetic analysis was conducted for each family members for disease diagnosis and genetic counselling.\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\n\u003ch2\u003ePedigree 2\u003c/h2\u003e\n\u003cp\u003eProband in pedigree 2 is a 30-years-old woman with four-year disease course. She had burning erythema and blisters on her face, neck and bilateral hand backs, with relief after sunlight avoidance (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. d-g). The symptoms were more severe in summer. The urine sample of the patient yielded pink fluorescent under Wood\u0026rsquo;s lamp (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. e). The patient was diagnosed as porphyria and suggested with light shielding. She received further genetic analysis because of the exaggerations of her skin symptoms in the past year. None of her family members had porphyria-related clinical manifestation, such as photodermatoses or acute neurovisceral attack.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eGenetic analysis\u003c/h2\u003e\n\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n\u003ch2\u003eWhole-exome sequencing\u003c/h2\u003e\n\u003cp\u003eWhole-exome sequencing of Pedigree 1 identified a novel deletion mutation with high pathogenicity: c.82_83delCC:p.Pro28* in proband 1 and her father; while sequencing in Pedigree 2 identified a intronic mutation of c.222\u0026thinsp;+\u0026thinsp;2delT in proband 2 and her father (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The pathogenicity was accessed according to guidance of The American College of Medical Genetics and Genomics (ACMG)(\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eresult of whole exome sequencing\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePedigree\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eVariant\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eMutation type\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePosition\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eZygote type\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePathogenicity\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eProband\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFather\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMother\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePedigree 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ec.82_83delCC:p.Pro28*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSmall deletion\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e161136714\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHeterozygote\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHeterozygote\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eExtremely highly pathogenic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePedigree 2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ec.222\u0026thinsp;+\u0026thinsp;2delT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSplicin\u003cspan class=\"Underline\"\u003eg\u003c/span\u003e mutation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e161137025\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHeterozygote\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHeterozygote\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eExtremely highly pathogenic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of a nonsense variant in\u003c/strong\u003e \u003cstrong\u003ePPOX\u003c/strong\u003e \u003cstrong\u003ein Pedigree 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSanger sequencing of the proband and her father indicated the presence of a heterozygous nonsense variant c.82_83delCC:p.Pro28* in \u003cem\u003ePPOX\u003c/em\u003e. This variant led to the substitution of codon CCU coding for proline to a terminal codon at position 28 of the amino acid sequence (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of an intronic variant in\u003c/strong\u003e \u003cstrong\u003ePPOX\u003c/strong\u003e \u003cstrong\u003ein Pedigree 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSanger sequencing of the proband and her father indicated the presence of a heterozygous intronic variant c.222\u0026thinsp;+\u0026thinsp;2delT in \u003cem\u003ePPOX\u003c/em\u003e. Because the position is very close to exon 2, it may lead to a different protein sequence by impacting the process of splicing.\u003c/p\u003e\n\u003cp\u003eIn order to verify the impact of variant c.222\u0026thinsp;+\u0026thinsp;2delT on splicing, rtPCR was conducted based on RNA of the proband 2, followed by amplification and Sanger sequencing of the exon 2 and exon 3 of the acquired cDNA. The result indicated an insertion of intron2 between these two exons (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003ePathogenicity analysis\u003c/h2\u003e\n\u003cp\u003eTo explore the conservatism of the mutation site in Pedigree 1, a conservation analysis was conducted by comparing protein sequence among different species, including human, mouse and cat. The result showed that the mutation site was highly conservative between different species (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. b). Protein structure of normal and mutated PPOX were predicted and the result showed that the small deletion in Pedigree 1 (c.82_83delCC) led to a premature termination of the transcription(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. c-d) and that the splicing mutation in Pedigree 2 (c.222\u0026thinsp;+\u0026thinsp;2delT) could cause the insertion of 45 amino acids (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. d-e) between exon2 and exon3.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis research identified two novel mutations of \u003cem\u003ePPOX\u003c/em\u003e. Variant in pedigree1 was a nonsense mutation(c.82_83delCC:p.Pro28*), leading to premature termination of the transcription and abnormal protein structure. Although variant at the site of first pedigree has not been reported before, previous research has identified p.Cys26* in VP pedigree(\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e), which is close to the mutation in pedigree 1 reported in this study. Apart from that, a frameshift variant c.78insC near the mutation site in pedigree 1 has been reported to be able to cut down the relative PPOX activity to 0% compared to wildtype in both eukaryotic and procaryotic expression systems （10）. Variant in pedigree 2 (c.222\u0026thinsp;+\u0026thinsp;2delT ) was a typical splicing mutation located at a highly conservative site in intron 2 of \u003cem\u003ePPOX\u003c/em\u003e gene. The variant could be pathogenic by impacting the process of normal splicing and protein structure and function. It is noteworthy that the in spite of the similar disease course, proband 1 tended to have more obvious hyperpigmentation and scar formation than proband 2. Furthermore, the liver function of proband 1 was extensively impaired, indicating a likely more severe phenotype associated with the nonsense mutation(c.82_83delCC:p.Pro28*), probably due to its huge impact on the protein structure(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. c-d). However, her father carrying the same mutation did not suffer from any porphyria attack, indicating the onset of the disease is not solely dependent on hereditary factors.\u003c/p\u003e\n\u003cp\u003eIn fact, VP has been reported to be an autosomal dominant inherited disease with low penetrance. Clinical presentation of VP varies from no overt clinical symptoms, chronic photosensitivity to acute neurovisceral crises. Penetrance of 40% and 50% has been reported in two pedigrees of VP carrying variant p. R59W and IVS6\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;A, respectively (\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e). Haplogroup H of mtDNA is also associated with the penetrance（12）. Up to now, over 180 mutations of VP have been identified （13）. It has been reported that among the three most common mutations in Finnish VP patients, the missense variant I12T might be an indicator for milder symptom （14）, however, there is no clear correlation between genotype and phenotype according to most of the previous publication（15, 16）. Even among individuals with the same variant, clinical manifestation could be different （17）, as in our presenting pedigrees.\u003c/p\u003e\n\u003cp\u003eOf the eight porphyria, five are inherited through an autosomal dominant pattern. In addition to VP, the clinical penetrance of other four AD porphyria is also low. Porphyria attacks might be induced by factors which either lead to increasing demand for heme or an additional decrease in enzyme activity(\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e). These non-hereditary factors include medications, infections, dieting, alcohol, hormones and neuropsychiatric factors（13）. It has been reported that exogenous oestrogen is associated with the onset of PCT （18）. Some scholars also believe that acute hepatic porphyria could be triggered by various hormonal changes, such as the use of high progesterone birth control medications（19, 20）. Furthermore, in AIP and VP, some of the acute attacks are related to menstruation, which could probably be reversed by Gonadotropin-releasing hormone (GnRH) agonists（20, 21）. Recently, a retrospective study has evaluated the penetrance of VP in 23 genetically heterogenous Finnish families and found that penetrance was higher among female than male (44% vs. 33%), and that during reproductive years(12\u0026ndash;49 years of age), the risk for acute symptoms is at the highest, reenforcing that hormone might be a contributing factor in the pathogenesis of the disease（22）.In this study, both female probands were heterozygotes who inherited their mutations from their heterozygous fathers who had not suffered from any symptoms related to VP yet. Besides, they both have overt clinical manifestation in their reproductive years. This could be possibly explained by their sexuality and hormone levels as indicated in previous publication.\u003c/p\u003e\n\u003cp\u003eIn this study, we identified two novel mutations of \u003cem\u003ePPOX\u003c/em\u003e gene: c.82_83delCC:p.Pro28* and c.222\u0026thinsp;+\u0026thinsp;2delT, which cause abnormal structure and function of the protein. The penetrance of these two mutations might be associated with sexuality. These findings expanded the genotypic and phenotypic spectrum of VP, which would be of critical value for disease diagnosis, genetic counseling and patient management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National High Level Hospital Clinical Research Funding (No. 2022-PUMCH-A-164 to T.W), the National College Students Innovation and Entrepreneurship Training Program (No.202010023059 to S.L,X.Z), the Beijing Natural Science Foundation (No. Z210017 to T.W) and Peking Union Medical College Hospital (No. ZC201911051 to T.W).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eD M Bissell, K E Anderson and H L Bonkovsky Porphyria. N Engl J Med 2017;377:862-872.\u003c/li\u003e\n\u003cli\u003eR E Kirsch, P N Meissner and R J Hift Variegate porphyria. Semin Liver Dis 1998;18:33-41.\u003c/li\u003e\n\u003cli\u003eP Mustajoki Variegate porphyria. Twelve years\u0026apos; experience in Finland. Q J Med 1980;49:191-203.\u003c/li\u003e\n\u003cli\u003eZ Karim, S Lyoumi, G Nicolas\u003cem\u003e, et al.\u003c/em\u003e Porphyrias: A 2015 update. Clin Res Hepatol Gastroenterol 2015;39:412-25.\u003c/li\u003e\n\u003cli\u003eA Luvai, W Mbagaya, D Narayanan\u003cem\u003e, et al.\u003c/em\u003e Hepatocellular carcinoma in variegate porphyria: a case report and literature review. Ann Clin Biochem 2015;52:407-12.\u003c/li\u003e\n\u003cli\u003eA Manzin, G Salvoni, P Bagnarelli\u003cem\u003e, et al.\u003c/em\u003e A single-step DNA extraction procedure for the detection of serum hepatitis B virus sequences by the polymerase chain reaction. J Virol Methods 1991;32:245-53.\u003c/li\u003e\n\u003cli\u003eD C Rio, M Ares, Jr., G J Hannon\u003cem\u003e, et al.\u003c/em\u003e Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc 2010;2010:pdb.prot5439.\u003c/li\u003e\n\u003cli\u003eS Richards, N Aziz, S Bale\u003cem\u003e, et al.\u003c/em\u003e Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405-24.\u003c/li\u003e\n\u003cli\u003eS D Whatley, N G Mason, J R Woolf\u003cem\u003e, et al.\u003c/em\u003e Diagnostic strategies for autosomal dominant acute porphyrias: retrospective analysis of 467 unrelated patients referred for mutational analysis of the HMBS, CPOX, or PPOX gene. Clin Chem 2009;55:1406-14.\u003c/li\u003e\n\u003cli\u003eM von und zu Fraunberg, R Tenhunen and R Kauppinen Expression and characterization of six mutations in the protoporphyrinogen oxidase gene among Finnish variegate porphyria patients. 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J Inherit Metab Dis 2005;28:277-86.\u003c/li\u003e\n\u003cli\u003eM Yasuda, B Chen and R J Desnick Recent advances on porphyria genetics: Inheritance, penetrance \u0026amp; molecular heterogeneity, including new modifying/causative genes. Mol Genet Metab 2019;128:320-331.\u003c/li\u003e\n\u003cli\u003eR J Hift and P N Meissner An analysis of 112 acute porphyric attacks in Cape Town, South Africa: Evidence that acute intermittent porphyria and variegate porphyria differ in susceptibility and severity. Medicine (Baltimore) 2005;84:48-60.\u003c/li\u003e\n\u003cli\u003eE Innala, T Backstrom, M Bixo\u003cem\u003e, et al.\u003c/em\u003e Evaluation of gonadotropin-releasing hormone agonist treatment for prevention of menstrual-related attacks in acute porphyria. Acta Obstet Gynecol Scand 2010;89:95-100.\u003c/li\u003e\n\u003cli\u003eK Baumann and R Kauppinen Penetrance and predictive value of genetic screening in acute porphyria. Mol Genet Metab 2020;130:87-99.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"variegate porphyria, mutation, protoporphyrinogen oxidase","lastPublishedDoi":"10.21203/rs.3.rs-3330892/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3330892/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eVariegate porphyria (VP) is an autosomal dominant inherited disease caused by mutation of the \u003cem\u003ePPOX\u003c/em\u003e gene and impacted activity of the protoporphyrinogen oxidase (PPOX). We report two pedigrees of young women who present VP-related manifestation such as blistering, flushing, scarring, and hyperpigmentation after sunlight exposure. The genetic analysis of \u003cem\u003ePPOX\u003c/em\u003e gene was performed.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWhole exome sequencing was conducted for both probands, followed by poly chain reaction (PCR) to amplify interested region and validate the suspicious mutations. Reverse transcriptive PCR was conducted to explore the impact of intronic mutation on the splicing process.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe genetic analysis revealed two novel mutations: c.82_83delCC:p.Pro28* and c.222\u0026thinsp;+\u0026thinsp;2delT in heterozygous state. The first mutation leads to a premature termination of \u003cem\u003ePPOX\u003c/em\u003e translation and the second one causes the insertion of intron2 between exon2 and exon3. Both of the probands inherit \u003cem\u003ePPOX\u003c/em\u003e mutation from their healthy father.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur study provides molecular diagnosis for two VP pedigrees and identified two novel \u003cem\u003ePPOX\u003c/em\u003e mutations. We propose a hypothesis that the incomplete penetrance of VP in these two cases might be associated with sexuality and hormone level.\u003c/p\u003e","manuscriptTitle":"Novel PPOX mutations in two variegate porphyria pedigrees","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-12 14:08:50","doi":"10.21203/rs.3.rs-3330892/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3b36efb8-e05f-4b41-8f30-a7a702307900","owner":[],"postedDate":"September 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-03T23:29:14+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-12 14:08:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3330892","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3330892","identity":"rs-3330892","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}